Type I diabetes (T1D) is a spontaneous disease in which cells of the immune system trigger inflammation in the pancreas leading to a specific destruction of the insulin-producing 2 cells within the pancreatic islets. CD4 T cells specific for glutamic acid decarboxylase (GAD) or insulin 2 chain (INS2) play a major role in the manifestation of T1D. Regulatory T cells (Tregs) are usually able to control the diabetogenic T cells and maintain tolerance. However, in disease prone mice, such as the non-obese diabetic (NOD) mouse, and in susceptible humans, genetic and environmental factors interfere with the development and/or function of Tregs, leading to activation of autoreactive T cells and manifestation of T1D. Recently, we discovered a novel population of T regulatory cells that expresses intermediate (int) levels of FoxP3, the signature transcription factor for Tregs, and high level of ROR3t, the signature transcription factor for Th17 cells. These cells, which we designate FoxP3int, also express CD62L, a molecule used for trafficking, and membrane-bound TGF2 which is utilized for suppression of effector T cells. FoxP3int T cells arise in the splenic T cell repertoire prior to disease development and migrate to the pancreas during insulitis. Ig-GAD1, an Ig molecule genetically engineered to express the diabetogenic 524-543 epitope of GAD, expands FoxP3int T cells without causing further differentiation. In fact, Ig-GAD1-expanded FoxP3int T cells migrate to the pancreas upon transfer into NOD.scid mice recipient of diabetogenic T cells and suppress the effector T cells while preserving dual expression of FoxP3int and ROR3t. The major hypothesis to be tested in this proposal postulates that a). FoxP3int T cells represent thymic derived FoxP3-expressing transitional intermediates that up-regulate ROR3t and mTGF2 in the spleen under non inflammatory encounters with self-antigen. b). the cells are readily expandable with Ig-GAD1 at the pre-insulitis stage and effectively migrate to the pancreas at the insulitis stage where they exercise broad suppressive function against diabetes. c). interaction with circulating effector T cells in the spleen leads to loss of mTGF2 and d). extended injection of higher doses of Ig-GAD1 will likely outcompete those interactions and expand FoxP3int T cells that could reverse T1D at advanced stages of the disease. Three aims are proposed to test this hypothesis. Aim one will test extended Ig-GAD1 regimens for expansion of FoxP3int Tregs at advanced stages of T1D. Also this aim will test FoxP3int T cells as a cellular therapy for advanced T1D diabetes and determine whether they originate from thymic natural Tregs. Aim 2 will define the role of antigen-specificity in the expansion and function of FoxP3int T cells and aim 3 will determine whether mTGF2 expression on FoxP3int T cells is regulated by effector T cells. Understanding how Tregs arise and suppress T1D could yield information useful for prevention and reversal of the disease. PUBLIC HEALTH RELEVANCE: The study proposed in this application will investigate antigen-specific mechanisms underlying expansion of regulatory T cells and suppression of type I diabetes. A novel population of FoxP3 regulatory T cells has been identified and understanding how these suppressive T cells function and interact with diabetogenic effector lymphocytes should yield information useful for the design of approaches to protect against the disease. Also, the approach takes advantage of an unique peptide delivery system that is adaptable for investigation of novel mechanistic aspects of Treg development and function. The study could impact the field of type I diabetes and autoimmunity both at the basic science and clinical levels.